The realization that nearly all cancers have mutations that disrupt cell cycle control led to the expectation that cell cycle-directed therapy, particulary against the cyclin-dependent kinases (CDKs), would revolutionize cancer therapy. However, while the promise of this approach is nearing fruition through the use of CDK4/6 inhibitors, it has been less successful with CDK2, a kinase with central roles in quiescence, DNA replication, and DNA damage. CDK2 is thought to mediate many oncogenic signaling pathways and because its activity is abnormally high in cancer, most therapeutic approaches seek to inhibit CDK2. Instead, we seek to utilize CDK2 activity as a therapeutic modality rather than as a target for inhibition. Normal cells tightly regulate CDK2, and its inhibitory phosphorylation by Wee1 is a particularly important control in the contexts of replication stress and DNA damage. Indeed, CDK2 inhibitory phosphorylation is essential for a successful replication stress response, and the lack thereof during S-phase arrest causes catastrophic replication stress failure and irreparable DNA damage. The goals of this proposal are to utilize novel models and approaches to understand the functions of CDK2 and its inhibitory phosphorylation, and to apply these insights to exploit CDK2-induced replication stress failure to kill cancer cells. Aim 1's objectiv is to gain new understanding into how inhibitory phosphorylation and CDK2 activity regulate replication stress, DNA damage, and tumorigenesis. The first subaims employ targeted CDK2 mutations in human cells to study known mechanisms that may link CDK2 inhibitory phosphorylation to replication stress failure. The next set of subaims focuses on new mechanisms connecting CDK2 to DNA damage and replication stress control. Finally, we will use endogenous CDK2AF knockin mice to determine the role of CDK2 inhibitory phosphorylation in tumor suppression. By combining physiologic human and murine models with biochemical and proteomic methods, we hope to make fundamental advances into CDK2's normal and neoplastic functions. Aim 2 will determine if CDK2-driven replication stress can be used to repurpose chemotherapy that transiently inhibits S-phase into agents that kill cancer cells. Several features of cancer cells suggest that induced replication stress failure may exhibit the required therapeutic index, including their abnormally high proliferative rates and CDK2 activity, and the presence of oncogenic mutations that exacerbate CDK2- driven replication stress failure. This aim has two objectives: 1) to identify the molecular features that render cancers most sensitive to this approach, and 2) to establish mouse models to optimize and validate the concept of harnessing CDK2-driven replication stress as chemotherapy. If successful, these studies will provide new insights into CDK2 and cancer, and form the basis for future trials of using CDK2-driven replication stress failure to treat cancer.